Protocols in Current Issue
    Tryptophan Fluorescence Quenching Assays for Measuring Protein-ligand Binding Affinities: Principles and a Practical Guide
    Authors:  Anthony Yammine, Jinlong Gao and Ann H. Kwan, date: 06/05/2019, view: 1951, Q&A: 0
    [Abstract] Tryptophan fluorescence quenching is a type of fluorescence spectroscopy used for binding assays. The assay relies on the ability to quench the intrinsic fluorescence of tryptophan residues within a protein that results from changes in the local environment polarity experienced by the tryptophan(s) upon the addition of a binding partner or ligand. ...
    In vitro Membrane Interaction and Liposome Fusion Assays Using Recombinant Hepatitis C Virus Envelope Protein E2
    [Abstract] In order to study the mechanism underlying the Hepatitis C Virus (HCV) fusion process we have performed assays using phospholipid liposomes and a truncated form of E2 protein, E2661 (amino acids 384-661 of the HCV polyprotein) lacking the transmembrane region. E2661 has been previously generated by using the baculovirus ...
    Preparation of Cell-free Synthesized Proteins Selectively Double Labeled for Single-molecule FRET Studies
    Authors:  Mayuri Sadoine, Michele Cerminara, Jörg Fitter and Alexandros Katranidis, date: 06/20/2018, view: 2422, Q&A: 0
    [Abstract] Single-molecule FRET (smFRET) is a powerful tool to investigate molecular structures and conformational changes of biological molecules. The technique requires protein samples that are site-specifically equipped with a pair of donor and acceptor fluorophores. Here, we present a detailed protocol for preparing double-labeled proteins for smFRET ...
    Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP
    Authors:  Tomoharu Kanie and Peter K. Jackson, date: 04/05/2018, view: 5349, Q&A: 0
    [Abstract] GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct ...
    Quantification of Colibactin-associated Genotoxicity in HeLa Cells by In Cell Western (ICW) Using γ-H2AX as a Marker
    Authors:  Sophie Tronnet and Eric Oswald, date: 03/20/2018, view: 2789, Q&A: 0
    [Abstract] The genotoxin colibactin is produced by several species of Enterobacteriaceae. This genotoxin induces DNA damage, cell cycle arrest, senescence and death in eukaryotic cells (Nougayrède et al., 2006; Taieb et al., 2016). Here we describe a method to quantify the genotoxicity of bacteria producing colibactin following a ...
    Measurement of Energy-dependent Rhodamine 6G Efflux in Yeast Species
    Authors:  Yvetta Gbelska, Nora Toth Hervay, Vladimira Dzugasova and Alexandra Konecna, date: 08/05/2017, view: 4217, Q&A: 0
    [Abstract] Rhodamine 6G is a highly fluorescent dye often used to determine the transport activity of yeast membrane efflux pumps. The ATP-binding cassette transporter KlPdr5p confers resistance to several unrelated drugs in Kluyveromyces lactis. KlPdr5p also extrudes rhodamine 6G (R6G) from intact yeast cells in an ...
    Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method
    Authors:  Karin Ernits, Katrin Viigand, Triinu Visnapuu, Kristina Põšnograjeva and Tiina Alamäe, date: 06/20/2017, view: 5207, Q&A: 0
    [Abstract] α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput differential scanning fluorometry method, also ...
    In vitro Microtubule Bundling Assay under Physiological Conditions
    Authors:  Zach Geisterfer, Gin Tezuka and Li Tao, date: 04/05/2017, view: 4751, Q&A: 0
    [Abstract] Kinesins play a role in organizing the mitotic spindle through the crosslinking of microtubules (MTs), made possible through binding sites at opposite ends of the holoenzyme. Here, we developed a method to test kinesin MT crosslinking action under physiological conditions.
    Gliding Assay to Analyze Microtubule-based Motor Protein Dynamics
    Authors:  Albert Shim, Gin Tezuka, Luke Kupcha and Li Tao, date: 04/05/2017, view: 7364, Q&A: 0
    [Abstract] The purpose of this protocol is to provide an updated method of performing microtubule gliding assays and visualizing it using fluorescence microscopy.
    Protein Synthesis Rate Assessment by Fluorescence Recovery after Photobleaching (FRAP)
    Authors:  Nikos Kourtis and Nektarios Tavernarakis, date: 03/05/2017, view: 5128, Q&A: 0
    [Abstract] Currently available biochemical methods cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. Here, we describe a non-invasive method for monitoring protein synthesis in single cells or tissues with intrinsically different translation rates, in live Caenorhabditis elegans animals.

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